Reversible genic male sterility in compositae

ABSTRACT

The present invention relates to plants of the Compositae family exhibiting a reversible genic male sterility trait, characterized in that the genic male sterility may be caused by a reduction or complete absence of endogenous jasmonic acid production, resulting from interference with one or more target genes involved in endogenous jasmonic acid production, selected from the group consisting of lipoxygenase, allene oxide synthase, allene oxide cyclase and 12-oxo-phytodienoic acid-10,11-reductase, or their functional homologs.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application claims benefit of European patent application SerialNo. 13151073.7 filed 11 Jan. 2013.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appln cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to Compositae plants exhibiting areversible genic male sterility trait. The invention further relates tocells, seeds and progeny of such plants, and to propagation material forobtaining the plants. The invention also relates to markers and the useof the markers for identifying the presence of the reversible genic malesterility trait.

BACKGROUND OF THE INVENTION

In commercial plant breeding the production of hybrid seed is veryimportant. Plants grown from hybrid seed are generally very uniform, andthey benefit from heterosis (hybrid vigour), which can lead to asignificant increase in yield and/or performance when compared to theparental lines of the hybrid, or to outcrossing (open-pollinated) lines.Typically the parental lines used for hybrid seed production are inbred,which implies that their genomes are largely homozygous. The combinationof two largely homozygous genomes into a hybrid leads to a high degreeof heterozygosity, if both parental lines were genetically unrelated ornot closely related.

Efficient hybrid seed production in plant species that are able toself-fertilise requires adequate measures to prevent self-fertilisationof the plants on which hybrid seeds are to be produced. Variousstrategies have been developed to achieve this, and to obtain anefficient hybrid seed production setup. However, the complexity andamount of labour required for each of these strategies varies greatly.

A strategy that naturally occurs in certain plant species is thephysical separation of male and female reproductive organs in separateflowers, either on separate plants (dioecious species) or on the sameplant (monoecious species). This system naturally promotes outcrossing,and it can be easily taken advantage of for hybrid seed production.

Another natural strategy is self-incompatibility, which has e.g. beenextensively studied in Brassica species. In this case, pollen isphysically unable to fertilise egg cells from the same plant. Theprecise mechanism of the incompatibility interaction can differ. Eitherpollen hydration or germination is prevented, or pollen tube growththrough the style is inhibited by the female tissues, or the pollen tubeis not attracted to ripe ovules, or the sperm nuclei are unable to mergewith the egg cell nucleus to form a viable zygote. Again, this naturallyoccurring system is very efficient and useful for preventingself-fertilisation, and for promoting outcrossing.

Another method for preventing selfing, which is typically used in e.g.maize, is the mechanical elimination of all male flowers (detasseling).The only flowers remaining on the plant are female, and these can bemanually pollinated with pollen from a selected paternal line, in orderto obtain ears with exclusively hybrid kernels.

In plant species with hermaphroditic flowers (producing both ovules andpollen grains within the same flower), a common strategy for preventingselfing is emasculation by mechanical removal of anthers and/or pollenprior to anthesis. When the anthers are mechanically removed before thepollen grains are released from the loculi and/or before the filamenthas extended far enough to match the height of the stigma, selfing isefficiently prevented. Subsequently the female reproductive parts of theemasculated flower are allowed to mature normally, after which pollengrains from a selected father plant can be deposited on the stigma, inorder to obtain exclusively hybrid seeds from the cross. Especially forcommercial-scale applications this method is however verylabour-intensive and not 100% reliable: if anthers are removed in aslightly too late developmental stadium or if one anther is accidentallynot removed, this can lead to a mixed seed set, consisting of hybrid andmaternal seeds. This results in non-uniformity of the commercial seedbatch, which is undesired for customers who expect uniform andconsistently superior seeds, and it brings inbred mother lines of hybridvarieties into commerce, which is undesired for the breeding company. A100% reliable hybrid system is therefore desirable.

Another approach is to induce male sterility by means of chemicals. Thisso-called male gametocide can be achieved by treatment with e.g.gibberellins (in rice and maize), sodium methyl arsenate (in rice), ormaleic acid (in wheat and onion). Disadvantages of this approach are thefact that this male sterility is not inheritable as it does not resultfrom a genetic determinant present in the plant's genome, and thatchemical treatment is labour intensive and not 100% reliable.

Another category of mechanisms through which selfing can be prevented,is termed genetic male sterility. Here three different approaches can bedistinguished: genetic-engineered male sterility (transgenic MS),cytoplasmic male sterility (CMS) and genic male sterility (GMS).Transgenic MS comprises all approaches that use a transgene to ensurethat pollen grains are unable to fertilise ovules, and that either leadto the death of pollen grains prior to anthesis, or to thedysfunctionality of pollen grains at anthesis. A well-known example isthe reversible Barnase/Barstar system, wherein the Barnase enzyme istransgenically expressed in the tapetum, which leads to pollensterility. However, when the Barstar protein is co-expressed in thetapetum, it blocks Barnase activity and restores pollen fertility(Mariani et al., 1990, Nature 347: 737-41).

Cytoplasmic male sterility (CMS) is a type of sterility that is undercontrol of extra-nuclear, cytoplasmic factors, more precisely of plastidorigin. Usually mutations in the mitochondrial genome underlie CMS, andthey typically inherit in a maternal fashion. Routine hybrid seedproduction with CMS lines requires the use of maintainer and restorerlines, which complicates the process and increases the costs and timerequired for commercial hybrid seed production.

Genic male sterility (GMS) encompasses a nuclear influence on malefertility, in contrast to cytoplasmic influences which are caused byorganellar factors. Due to e.g. a mutation in a nuclear gene the plantdoes not produce viable and/or functional pollen grains or male spores,and/or it is unable to disperse its pollen due to e.g. non-dehiscence ofits anthers.

The Asteraceae family—also known as the Compositae family—is one of thelargest extant plant families. It comprises various commerciallyimportant crop plants, such as sunflower (Helianthus), lettuce(Lactuca), endive, witloof and radicchio (Cichorium), artichoke(Cynara), and many ornamental plants such as Chrysanthemum, Tagetes,Gerbera and Zinnia.

In the Compositae family only few types of male sterility have beendeveloped for commercial exploitation and hybrid seed production.Emasculation is very difficult in this family, due to the compositenature of the inflorescences. In e.g. endive and witloof,self-pollination is normally prevented by spraying an inflorescence withwater, to flush away the pollen. Timing is crucial, because thisspraying needs to be done immediately after opening of the anthers,before the stigma splits into two curving parts that protrude beyond theanthers. After spraying the inflorescence has to be blown dry and has tobe allowed to develop further, before pollen grains from a selectedfather can be deposited onto the stigma. It is critical to choose theoptimal moment for spraying with water: if pollen are removed too latesome self-pollination will already have occurred, and if pollen areremoved before the anthers are fully opened, pollen grains will remainpresent and may cause self-pollination at a later stage. Every day thereis only a limited time window during which this spraying can be done,and its timing depends strongly on light and temperature conditions. Thedevelopment of an efficient, reversible male sterility system in theCompositae family would thus greatly facilitate breeding in crop speciesbelonging to this family.

Hybrid sunflowers are available and can be produced using variousmethods described above. However for e.g. endive a hybrid system is notyet available at all, and a major problem is the very limited geneticvariation within this cultivated crop. If an efficient hybrid systemwould be available for endive, this could be used to increase thegenetic variation, as well as provide additional benefits throughheterotic effects. Interspecific crosses with e.g. witloof (Cichoriumintybus) are possible, and these could be used to introduce foreigngenetic material into endive germplasm, but this would lead tocomplications at the genetic level, namely the fact that the resultingprogeny would no longer be purely endive, but a mixture of endive andwitloof.

In witloof a recessive GMS trait has previously been created through atransposon insertion in a homologue of the DYT1 gene of Arabidopsis(Quillet et al., 2011, Cloning and characterization of nuclear malesterility 1 (nms1) in chicory. However, in practice it is very difficultto transfer this transposon-based trait to endive (Cichorium endivia).Also, although the transposon may spontaneously be excised from the DYT1gene homologue and thus potentially restore fertility, a researcher isnot able to easily, predictably and consistently reverse this malesterility trait in witloof whenever he wishes to do so.

CMS has also been created in witloof, by combining the nuclear genomefrom C. intybus with cytoplasm from sunflower (Helianthus annuus).However, again this is not a reversible male sterility, and theperpetuation of the trait requires maintainer and restorer lines.

In lettuce, male sterility has been described in the prior art, andpollination for obtaining hybrid seeds can e.g. be achieved with bees(U.S. Pat. No. 7,569,743). Dominant GMS is available (resulting from theMS7 mutation), as well as CMS (through combination of the nuclear genomeof Lactuca sativa with the cytoplasm of sunflower). However, none ofthese traits is reversible, and it thus requires more efforts tomaintain the male sterile mother lines in any of those cases.

One could imagine a transgenic approach to obtain reversible malesterility in Compositae plants, e.g. with the Barnase/Barstar system,but such transgenic plants would have a “regulated” status, which isundesired in e.g. the European market, and this would necessitate largeextra deregulation expenses to obtain market approval. The cost forbringing hybrid seeds resulting from the use of such a transgenicreversible male-sterile Compositae plant to the market would thus becomequite high.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide areversible male sterility system in Compositae plants for the efficientand convenient production of hybrid seeds.

In the research leading to the present invention it was found thatCompositae plants may be rendered male-sterile through aloss-of-function mutation in the OPR3 gene, and that this male sterilitymay be reversed by the application of methyl jasmonate (MeJA) and/orjasmonic acid or other jasmonic acid derivatives to flower buds.

The OPR3 gene encodes the 12-oxophytodienoic acid reductase protein,which is a key enzyme in the biosynthesis of the phytohormone jasmonicacid. In the model plant species Arabidopsis thaliana jasmonic acid isrequired for male fertility, and mutations in the Arabidopsis OPR3 genewere reported to cause male sterility. Male fertility could be restoredby treating flower buds with methyl jasmonate (Stintzi and Browse, 2000;Proc. Natl. Acad. Sci. USA 97: 10625-10630). However, this observationin Arabidopsis is apparently not by definition valid for other plantspecies. In tomato, for example, jasmonic acid is required for thematernal control of seed maturation, but not for male fertility (Li etal., 2004; Plant Cell 16: 126-143). This indicates that the jasmonicacid pathway regulates distinct developmental processes in differentplant species.

It is surprising that targeting functional homologues of the OPR3 genein a plant family other than the Brassicaceae actually leads toreversible male sterility, especially since from a phylogenetic point ofview the Compositae family is closer related to the Solanaceae family(comprising tomato, in which jasmonic acid is not involved in malefertility) than to the Brassicaceae family (comprising Arabidopsis).Among the eudicots, the Compositae and Solanaceae families both belongto the Asterids Glade, whereas the Brassicaceae family belongs to theRosids Glade. The skilled person would not be able to establish, withoutundue burden, in which plant families other than the Brassicaceae orSolanaceae, which have been investigated in the prior art, the jasmonicacid pathway may or may not regulate male fertility.

Reversible genic male sterility provides a great advantage overnon-reversible genic male sterility. When the genic male sterility traitperforms in a reliable and consistent manner, a male-sterile motherplant will exclusively produce hybrid seeds, when pollinated with pollenfrom a selected father plant (e.g. through manual pollination or insectpollination), without a need for emasculation or other means to inducemale sterility. In addition, it is very easy to propagate and maintainthe male sterile mother plant: upon a specific treatment, the malesterility may be reversed and the mother plant becomes entirely fertile,and it may thus self-fertilise to produce inbred seeds that arehomozygous for the said genic male sterility trait. In cases ofnon-reversible genic male sterility this is not possible, and it is moredifficult to maintain the genic male sterility trait from generation togeneration.

Accordingly, it is an object of the invention to not encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

Deposits

The Deposits with NCIMB, under deposit accession numbers 42060 and 42092were made pursuant to the terms of the Budapest Treaty. Upon issuance ofa patent, all restrictions upon the deposit will be removed, and thedeposit is intended to meet the requirements of 37 CFR §§1.801-1.809.The deposit will be maintained in the depository for a period of 30years, or 5 years after the last request, or for the effective life ofthe patent, whichever is longer, and will be replaced if necessaryduring that period.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings.

FIG. 1: Reversible male sterility in lettuce Panel A shows aninflorescence from a wild-type lettuce plant after flowering (left-handside), as compared to an inflorescence from a lettuce plant of theinvention after flowering (right-hand side). Neither of the plants hasbeen pollinated with pollen from another lettuce plant. The wild-typelettuce plant is fully fertile, and it has produced seeds throughselfing. The lettuce plant of the invention, in contrast, which has notbeen treated with jasmonic acid and/or one or more jasmonic acidderivatives, shows a complete absence of seeds in the inflorescence.This demonstrates that the presence of the reversible genic malesterility trait of the invention makes the plant completelyself-sterile. Panel B illustrates the effect of treatment with jasmonicacid and/or jasmonic acid derivatives on the fertility of a reversiblegenic male sterile lettuce plant of the invention. The inflorescence wastreated with methyl jasmonate (“MeJA”) during its development, and hasproduced seeds through selfing.

FIG. 2: Reversible male sterility in endive. Panel A shows the flowerand stamens of a wild-type endive plant (upper 2 pictures), which has ablue colour and normal pollen production, as compared to a typicalflower of a reversible genic male sterile endive plant of the invention,which has a pale flower colour and less pollen production (lower 2pictures). The pollen grains produced by the endive plants of theinvention were not functional. Panel B illustrates the effect oftreatment with jasmonic acid and/or jasmonic acid derivatives on thefertility of a reversible genic male sterile endive plant of theinvention. The left-hand picture shows an untreated flower of a plant ofthe invention, which is white in colour and male-sterile. The right-handpicture shows a plant of the invention with in the centre a flower thathas been treated with methyl jasmonate at an early stage of itsdevelopment. Its colour has changed to blue, and it has becomemale-fertile.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a plant of the Compositae family (also knownas the Asteraceae family) exhibiting a reversible genic male sterilitytrait, characterised in that the genic male sterility trait is caused bythe reduction or complete absence of endogenous jasmonic acidproduction, resulting from interference with one or more target genesinvolved in endogenous jasmonic acid production, selected from the groupconsisting of lipoxygenase, allene oxide synthase, allene oxide cyclaseand 12-oxo-phytodienoic acid-10,11-reductase, or their functionalhomologues.

The aforementioned genes encode the enzymes that constitute the jasmonicacid biosynthetic pathway in plants. Lipoxygenase converts α-linolenicacid to 13-hydroperoxylinoleic acid, which is further converted to12,13-epoxyoctadecatrienoic acid by the action of allene oxide synthase.The latter molecule is the substrate of allene oxide cyclase, by whichit is converted into (9S,13S)-12-oxo-phytodienoic acid, which may befurther processed into 3-oxo-2(2′[Z]-pentenyl)-cyclopentane-1-octanoicacid by the enzymatic action of the 12-oxo-phytodienoicacid-10,11-reductase (OPR3) enzyme. Three cycles of β-oxidation aresubsequently required to convert the3-oxo-2(2′[Z]-pentenyl)-cyclopentane-1-octanoic acid molecule into (3R,7S)-jasmonic acid.

In one embodiment the male sterility in plants of the invention may bereversed by the application of one or more jasmonic acid derivatives, inparticular methyl jasmonate. Methyl jasmonate (MeJA) is a volatilederivative of jasmonic acid from which it may be converted in planta ina reaction catalysed by the S-adenosyl-L-methionine:jasmonic acidcarboxyl methyltransferase enzyme.

In another embodiment jasmonic acid may be used.

Conditional and controlled reversibility of the male sterility phenotypeis very useful, because it allows the easy maintenance of male sterileplants of the invention across successive generations. The applicationof one or more jasmonic acid derivatives, in particular methyljasmonate, renders the male sterile plants of the invention entirelyfertile (both male- and female-fertile), and capable ofself-fertilisation. In this manner the male sterility trait of theinvention may be conveniently transmitted to the next generation,without the need for e.g. restorer or maintainer lines.

The application of jasmonic acid and/or one or more jasmonic acidderivatives, in particular methyl jasmonate, may be done in variousways, such as spraying them in an aqueous solution or directly applyingthem with a brush onto young flower buds in an aqueous solution, or on aregular basis adding them to the water or substrate provided to theplants, such that they are taken up by the plant and distributed thereinin a systemic manner.

In one embodiment the interfering with the one or more target genesinvolved in endogenous jasmonic acid production consists of preventingtranscription thereof. Preventing transcription may for example beachieved by means of RNA oligonucleotides, DNA oligonucleotides or RNAimolecules directed against the target gene promoter, or preferably bymeans of the expression of a negatively acting transcription factoracting on the target gene promoter.

In another embodiment the interfering with the one or more target genesinvolved in endogenous jasmonic acid production consists ofdestabilising the target gene mRNA or transcript, preferably by means ofnucleic acid molecules that are complementary to the target gene mRNA ortranscript, selected from the group consisting of antisense RNA, RNAimolecules, Virus-Induced Gene Silencing (VIGS) molecules, co-suppressormolecules, RNA oligonucleotides or DNA oligonucleotides.

In another embodiment the interfering with the one or more target genesinvolved in endogenous jasmonic acid production consists of inhibitingthe target gene expression product, preferably by means of theexpression product(s) of one or more dominant negative nucleic acidconstructs, or preferably by means of one or more chemical compounds.

In yet another embodiment the interfering with the one or more targetgenes involved in endogenous jasmonic acid production consists of theintroduction of one or more mutations into the target gene, leading toperturbation of its biological function. The one or more mutations arepreferably introduced randomly by means of one or more chemicalcompounds, such as ethyl methanesulphonate, nitrosomethylurea,hydroxylamine, proflavine, N-methyl-N-nitrosoguanidine,N-ethyl-N-nitrosourea, N-methyl-N-nitro-nitrosoguanidine, diethylsulphate, ethylene imine, sodium azide, formaline, urethane, phenol andethylene oxide, and/or by physical means, such as UV-irradiation,fast-neutron exposure, X-rays, gamma irradiation, and/or by insertion ofgenetic elements, such as transposons, T-DNA, retroviral elements. Theone or more mutations may also be introduced specifically by means ofhomologous recombination or oligonucleotide-based mutation induction.

This invention also relates to plants of the genus Lactuca, exhibiting areversible genic male sterility trait according to the presentinvention. In one embodiment, the plants belong to the species Lactucasativa, and the reversible male sterility trait is caused by a geneticdeterminant, the presence of which genetic determinant may be identifiedby a molecular marker characterised by SEQ ID No. 1, and wherein thesaid genetic determinant is as present in the genome of plants grownfrom seed of which a representative sample was deposited with the NCIMBunder accession number NCIMB 42060.

This invention also relates to Lactuca plants exhibiting a reversiblegenic male sterility trait, obtainable by crossing a first Lactuca plantwith a second Lactuca plant, wherein one of the said plants is grownfrom seeds of which a representative sample was deposited with the NCIMBunder accession number NCIMB 42060, or a progeny plant thereof, andselecting, preferably in the F2 generation, for plants that exhibit areversible genic male sterility trait.

Alternatively, selection may already be done in the F1 generation bymeans of a molecular marker detecting the SNP of SEQ ID No. 1.

This invention further relates to seed of a Lactuca plant exhibiting areversible genic male sterility trait, wherein the plant that may begrown from the seed may comprise the genetic determinant that may beidentified by a molecular marker characterised by SEQ ID No. 1, andwherein the said genetic determinant is as present in the genome ofplants grown from seed of which a representative sample was depositedunder accession number NCIMB 42060.

This invention further relates to progeny of a Lactuca plant exhibitinga reversible genic male sterility trait, which may comprise the geneticdeterminant that may be identified by a molecular marker characterisedby SEQ ID No. 1, and wherein the said genetic determinant is as presentin the genome of plants grown from seed of which a representative samplewas deposited under accession number NCIMB 42060.

Such progeny may be produced by sexual or vegetative reproduction of aplant of the invention or a progeny plant thereof. The progeny plantdisplays the reversible genic male sterility trait in the same or in asimilar way as the plant of which representative seed was depositedunder accession number NCIMB 42060. This means that such progeny has thesame characteristics as claimed for lettuce plants of the invention.

As used herein, the word “progeny” is intended to mean the offspring orthe first and all further descendants from a cross with a plant of theinvention that shows the reversible genic male sterility trait. Progenyof the invention may comprise descendants of any cross with a plant ofthe invention that carries the reversible genic male sterility trait.Such progeny is for example obtainable by crossing a first lettuce plantwith a second lettuce plant, wherein one of the lettuce plants was grownfrom seeds of a plant of the invention, representative seeds of whichwere deposited under NCIMB accession number 42060, but it may also bethe progeny of any other lettuce plant carrying the reversible genicmale sterility trait as present in seeds of deposit NCIMB 42060.

It is understood that a parent plant that provides the trait of theinvention is not necessarily a plant grown directly from the depositedseeds. The parent may also be a progeny plant from the seed, or aprogeny plant from seeds that are identified to have (or to haveacquired) the trait of the invention by other means. In one embodiment,the invention relates to lettuce plants that carry the trait of theinvention and that have acquired the said trait by introduction of thegenetic information that is responsible for the trait from a suitablesource, either by conventional breeding, or genetic modification, inparticular by cis-genesis or transgenesis. Cis-genesis is geneticmodification of plants with a natural gene, encoding an (agricultural)trait from the crop plant itself or from a sexually compatible donorplant. Transgenesis is genetic modification of a plant with a gene froma non-crossable species or with a synthetic gene.

In one embodiment, the source from which the genetic information isacquired is formed by plants grown from the deposited seeds, or bysexual or vegetative descendants therefrom.

“Progeny” also encompasses plants that carry the trait of the inventionwhich are obtained from other plants of the invention by vegetativepropagation or multiplication.

This invention also relates to propagation material suitable forproducing a Lactuca plant exhibiting a reversible genic male sterilitytrait, which may comprise the genetic determinant that may be identifiedby a molecular marker characterised by SEQ ID No. 1, and wherein thesaid genetic determinant is as present in the genome of plants grownfrom seed of which a representative sample was deposited under accessionnumber NCIMB 42060.

In one embodiment, the propagation material is suitable for sexualreproduction. Such propagation material may comprise for examplemicrospores, pollen, ovaries, ovules, embryo sacs and egg cells. Inanother embodiment, the propagation material is suitable for vegetativereproduction. Such propagation material may comprise for examplecuttings, roots, stems, cells, protoplasts, and tissue cultures ofregenerable cells, parts of the plant that are suitable for preparingtissue cultures, in particular leaves, pollen, embryos, cotyledons,hypocotyls, meristematic cells, root tips, anthers, flowers, seeds andstems.

The invention further relates to a lettuce plant grown or regeneratedfrom the said propagation material of a plant of the invention, whichplant exhibits the reversible genic male sterility trait of theinvention.

The invention further relates to a cell of a lettuce plant of theinvention, which cell may comprise a genetic determinant which leads toreversible genic male sterility, wherein the said genetic determinant isas present in a lettuce plant, representative seeds of which weredeposited under NCIMB accession number 42060. The said cell thus maycomprise the genetic information encoding the said reversible genic malesterility trait, in particular genetic information which issubstantially identical, preferably completely identical to the geneticinformation encoding the said reversible genic male sterility trait ofthe lettuce plant, representative seeds of which were deposited underNCIMB accession number 42060. Preferably, the cell of the invention ispart of a plant or plant part, but the cell may also be in isolatedform.

The invention also relates to a cell of a lettuce plant of theinvention, which cell may comprise a genetic determinant which leads toreversible genic male sterility, and which plant is obtained bytransferring the reversible genic male sterility trait as found in seedsthat were deposited under NCIMB accession number 42060 into anagronomically valuable lettuce plant.

The invention further relates to seed of the lettuce plant of theinvention, which seed contain in their genome the genetic informationthat encodes the reversible genic male sterility trait of the invention.

The invention also relates to the use of seeds that were deposited underNCIMB accession number 42060 for transferring reversible genic malesterility into another agronomically valuable lettuce plant.

The invention also relates to the use of a lettuce plant of theinvention that exhibits reversible genic male sterility due to thepresence, in the genome of the plant, of the reversible genic malesterility trait as found in seeds that were deposited under NCIMBaccession number 42060 as a crop.

The invention further relates to the use of a lettuce plant of theinvention that exhibits reversible genic male sterility due to thepresence, in the genome of the plant, of the reversible genic malesterility trait as found in seeds that were deposited under NCIMBaccession number 42060 as a source of seed.

The invention also relates to the use of a lettuce plant of theinvention that exhibits reversible genic male sterility due to thepresence, in the genome of the plant, of the reversible genic malesterility trait as found in seeds that were deposited under NCIMBaccession number 42060 as a source of propagating material.

The invention also relates to the use of a lettuce plant of theinvention that exhibits reversible genic male sterility due to thepresence, in the genome of the plant, of the reversible genic malesterility trait as found in seeds that were deposited under NCIMBaccession number 42060 for consumption.

The invention also relates to harvested leaves of lettuce plants of theinvention, to food products which may comprise harvested leaves oflettuce plants of the invention, either in natural or in processed form,and to a container which may comprise one or more lettuce plants of theinvention in a growth substrate for harvest of leaves from the lettuceplant in a domestic environment.

The invention further relates to the use of a lettuce plant of theinvention in breeding to confer reversible genic male sterility.

The invention also relates to the use of the reversible genic malesterility trait as found in seeds that were deposited under NCIMBaccession number 42060 for conferring reversible genic male sterilityonto a Lactuca sativa plant.

The invention further relates to the use of a Lactuca sativa plant as arecipient of the reversible genic male sterility trait as found in seedsthat were deposited under NCIMB accession number 42060.

In one aspect the invention relates to a method for production of aLactuca sativa plant which exhibits a reversible genic male sterilitytrait, which may comprise

-   -   a) crossing a plant which may comprise a genetic determinant        that leads to the trait with another plant;    -   b) selfing the resulting F1 for obtaining F2 plants;    -   c) selecting plants that have the trait in the F2;    -   d) optionally performing one or more additional rounds of        selfing or crossing, and subsequently selecting for a plant        which may comprise/showing the trait of the invention.

In one aspect, the invention relates to a method for production of aLactuca sativa plant which exhibits a reversible genic male sterilitytrait, which may comprise

-   -   a) crossing a plant which may comprise the genetic determinant        that leads to the trait with another plant;    -   b) optionally backcrossing the resulting F1 with the preferred        parent;    -   c) selecting for plants that have the trait in the F2;    -   d) optionally performing one or more additional rounds of        selfing or crossing, and subsequently selecting for a plant        which may comprise the trait.

The invention additionally provides a method of introducing anotherdesired trait into a Lactuca sativa plant which exhibits the reversiblegenic male sterility trait, which may comprise:

-   -   a) crossing a Lactuca sativa plant that exhibits the reversible        genic male sterility trait, representative seed of which were        deposited under deposit number NCIMB 42060, with a second        Lactuca sativa plant that may comprise a desired trait to        produce F1 progeny;    -   b) selecting an F1 progeny that may comprise said reversible        genic male sterility trait and the desired trait;    -   c) crossing the selected F1 progeny with either parent, to        produce backcross progeny;    -   d) selecting backcross progeny which may comprise the desired        trait and the reversible genic male sterility trait; and    -   e) optionally repeating steps c) and d) one or more times in        succession to produce selected fourth or higher backcross        progeny that may comprise the desired trait and the reversible        genic male sterility trait. The invention includes a Lactuca        sativa plant produced by this method.

In one embodiment selection for plants exhibiting the reversible genicmale sterility trait is done in the F1 or any further generation byusing a molecular marker characterised by SEQ ID No. 1. In anotheraspect selection for the trait of the invention is started in the F2 ofa cross or alternatively of a backcross. Selection of plants in the F2may be done phenotypically as well as by using the said marker whichdirectly or indirectly detects the genetic determinant underlying thetrait.

In one embodiment selection for plants exhibiting the reversible genicmale sterility trait is started in the F3 or a later generation.

In one embodiment the plant which may comprise the genetic determinantis a plant of an inbred line, a hybrid, a doubled haploid, or of asegregating population.

The invention further provides a method for the production of a Lactucasativa plant exhibiting the reversible genic male sterility trait byusing a doubled haploid generation technique to generate a doubledhaploid line which may comprise the said trait.

The invention also relates to a method for the production of a Lactucasativa plant exhibiting the reversible genic male sterility trait byusing a seed that may comprise a genetic determinant in its genome thatleads to the reversible genic male sterility trait for growing the saidLactuca sativa plant. The seeds are suitably seeds of which arepresentative sample was deposited under deposit number NCIMB 42060.

The invention also relates to a method for seed production which maycomprise growing Lactuca sativa plants from seeds of which arepresentative sample was deposited under deposit accession number NCIMB42060, reversing the reversible genic male sterility by the applicationof jasmonic acid and/or one or more jasmonic acid derivatives, inparticular methyl jasmonate, allowing the plants to produce seeds, andharvesting those seeds. Production of the seeds is suitably done bycrossing or selfing. Crossing may e.g. be done by means of handpollination or by employing suitable pollinating insects.

In one embodiment, the invention relates to a method for the productionof a Lactuca sativa plant exhibiting the reversible genic male sterilitytrait by using tissue culture.

The invention furthermore relates to a method for the production of aLactuca sativa plant exhibiting the reversible genic male sterilitytrait by using vegetative reproduction.

In one embodiment, the invention relates to a method for the productionof a Lactuca sativa plant exhibiting the reversible genic male sterilitytrait by using a method for genetic modification to introduce inparticular dominantly acting transgenes that cause the said trait intothe Lactuca sativa plant, for example by means of RNAi, amiRNA orantisense, or dominant-negative versions of a target gene, or throughmutation. Genetic modification may comprise transgenic modification ortransgenesis, using a gene from a non-crossable species or a syntheticgene, and cisgenic modification or cisgenesis, using a natural gene,coding for an (agricultural) trait, from the crop plant itself or from asexually compatible donor plant. The plant may also be geneticallymodified by mutation of a target gene to exhibit the male sterilitytrait of the invention.

The invention also relates to a breeding method for the development ofLactuca sativa plants that exhibit the reversible genic male sterilitytrait wherein germplasm which may comprise said trait is used.Representative seed of said plant which may comprise the geneticdeterminant and being representative for the germplasm was depositedunder deposit accession number NCIMB 42060.

In a further embodiment the invention relates to a method for theproduction of a Lactuca sativa plant exhibiting the reversible genicmale sterility trait wherein progeny or propagation material of a plantwhich may comprise the genetic determinant conferring said trait is usedas a source to introgress the said trait into another Lactuca sativaplant. Representative seed of said plant which may comprise the geneticdeterminant was deposited under deposit accession number NCIMB 42060.

The invention provides preferably a Lactuca sativa plant exhibiting thereversible genic male sterility trait, which plant is obtainable by anyof the methods herein described and/or familiar to the skilled person.

This invention also relates to the use of Lactuca sativa plantsexhibiting a reversible genic male sterility trait as a female parentfor the creation of hybrid Lactuca sativa seeds. The fact that Lactucasativa plants of the invention are unable to self-fertilise due to malesterility (if they are not rendered male fertile by the application ofone or more jasmonic acid derivatives, in particular methyl jasmonate)makes it possible to efficiently use Lactuca sativa plants of theinvention as female parents for the efficient creation of hybrid Lactucasativa plants, using pollen from other Lactuca sativa plants.Pollination of the plants of the invention may e.g. be achieved by meansof manual pollination, or by employing suitable pollinating insects,such as bumblebees, bees or flies and placing reversible genic malesterile Lactuca sativa plants of the invention alongside male-fertileLactuca sativa plants that will function as father plants of the hybridLactuca sativa seeds, by providing functional pollen grains for asuccessful fertilisation of the reversible genic male sterile Lactucasativa plants of the invention. This invention further relates to hybridlettuce seeds, obtainable by the use of Lactuca sativa plants exhibitinga reversible genic male sterility trait as a female parent.

According to a further aspect thereof the invention also relates toplants of the genus Cichorium, exhibiting the reversible genic malesterility trait. The genus Cichorium may comprise economically andagriculturally relevant crop species, such as Cichorium endivia (endive)and Cichorium intybus (witloof, radicchio), and also Cichorium spinosumand Cichorium pumillum, which may all be crossed to each other.

In one embodiment, the said plants belong to the genus Cichorium and inparticular to the species Cichorium endivia, and the reversible malesterility trait is caused by a genetic determinant, which is as presentin the genome of plants grown from seed of which a representative samplewas deposited under accession number NCIMB 42092, in which the presenceof the genetic determinant may be identified by a molecular markercharacterised by SEQ ID No. 2.

This invention also relates to Cichorium plants, in particular Cichoriumendivia plants, exhibiting a reversible genic male sterility trait,obtainable by crossing a first Cichorium plant, in particular aCichorium endivia plant, with a second Cichorium plant, in particular aCichorium endivia plant, wherein one of the said plants is grown fromseeds of which a representative sample was deposited with the NCIMBunder accession number NCIMB 42092, or a progeny plant thereof, andselecting, preferably in the F2 generation, for plants that exhibit areversible genic male sterility trait.

This invention further relates to seed of a Cichorium plant, inparticular a Cichorium endivia plant exhibiting a reversible genic malesterility trait, which is as present in the genome of plants grown fromseed of which a representative sample was deposited under accessionnumber NCIMB 42092, in which the presence of the genetic determinant maybe identified by a molecular marker characterised by SEQ ID No. 2.

This invention further relates to progeny of a Cichorium plant, inparticular a Cichorium endivia plant, exhibiting a reversible genic malesterility trait, which may comprise the genetic determinant, which is aspresent in the genome of plants grown from seed of which arepresentative sample was deposited under accession number NCIMB 42092,in which the presence of the genetic determinant may be identified by amolecular marker characterised by SEQ ID No. 2.

Such progeny may be produced by sexual or vegetative reproduction of aplant of the invention or a progeny plant thereof. The progeny plantdisplays the reversible genic male sterility trait in the same or in asimilar way as the plant of which representative seed was deposited(NCIMB 42092). This means that such progeny has the same characteristicsas claimed for endive plants of the invention.

As used herein the word “progeny” is intended to mean the offspring orthe first and all further descendants from a cross with a plant of theinvention that shows the reversible genic male sterility trait. Progenyof the invention may comprise descendants of any cross with a plant ofthe invention that carries the reversible genic male sterility trait.Such progeny is for example obtainable by crossing a first endive plantwith a second endive plant, wherein one of the endive plants was grownfrom seeds of a plant of the invention, representative seeds of whichwere deposited under NCIMB accession number 42092, but it may also bethe progeny of any other endive plant or another Cichorium plantcarrying the reversible genic male sterility trait as present in NCIMB42092.

It is to be understood that a parent plant that provides the trait ofthe invention is not necessarily a plant grown directly from thedeposited seeds. The parent may also be a progeny plant from the seed,or a progeny plant from seeds that are identified to have (or to haveacquired) the trait of the invention by other means. In one embodiment,the invention relates to endive plants that carry the trait of theinvention and that have acquired the said trait by introduction of thegenetic information that is responsible for the trait from a suitablesource, either by conventional breeding, or genetic modification, inparticular by cis-genesis or transgenesis. Cis-genesis is geneticmodification of plants with a natural gene, encoding an (agricultural)trait from the crop plant itself or from a sexually compatible donorplant. Transgenesis is genetic modification of a plant with a gene froma non-crossable species or with a synthetic gene.

In one embodiment, the source from which the genetic information isacquired is formed by plants grown from the deposited seeds, or bysexual or vegetative descendants therefrom. “Progeny” also encompassesplants that carry the trait of the invention which are obtained fromother plants of the invention by vegetative propagation ormultiplication.

This invention also relates to propagation material suitable forproducing a Cichorium plant, in particular a Cichorium endivia plantexhibiting a reversible genic male sterility trait, which may comprise agenetic determinant which is as present in the genome of plants grownfrom seed of which a representative sample was deposited under accessionnumber NCIMB 42092, in which the genetic determinant may be identifiedby a molecular marker characterised by SEQ ID No. 2.

In one embodiment, the propagation material is suitable for sexualreproduction. Such propagation material may comprise for examplemicrospores, pollen, ovaries, ovules, embryo sacs and egg cells. Inanother embodiment, the propagation material is suitable for vegetativereproduction. Such propagation material may comprise for examplecuttings, roots, stems, cells, protoplasts, and tissue cultures ofregenerable cells, parts of the plant that are suitable for preparingtissue cultures, in particular leaves, pollen, embryos, cotyledons,hypocotyls, meristematic cells, root tips, anthers, flowers, seeds andstems.

The invention further relates to a Cichorium plant grown or regeneratedfrom the said propagation material of a plant of the invention, whichplant exhibiting the reversible genic male sterility trait of theinvention.

The invention further relates to a cell of an endive plant of theinvention, which cell may comprise a genetic determinant which leads toreversible genic male sterility, wherein the said genetic determinant isas present in an endive plant, representative seeds of which weredeposited under NCIMB accession number 42092, in which the geneticdeterminant may be identified by a molecular marker characterised by SEQID No. 2. The said cell thus may comprise the genetic informationencoding the said reversible genic male sterility trait, in particulargenetic information which is substantially identical, preferablycompletely identical to the genetic information encoding the saidreversible genic male sterility trait of the endive plant,representative seeds of which were deposited under NCIMB accessionnumber 42092, in which the genetic determinant may be identified by amolecular marker characterised by SEQ ID No. 2. Preferably, the cell ofthe invention is part of a plant or plant part, but the cell may also bein isolated form.

The invention also relates to a cell of a Cichorium plant, in particulara Cichorium endivia plant of the invention, which cell may comprise agenetic determinant which leads to reversible genic male sterility, andwhich plant is obtained by transferring the reversible genic malesterility trait as found in seeds that were deposited under NCIMBaccession number 42092 into an agronomically valuable endive plant.

The invention further relates to seed of a Cichorium plant, inparticular a Cichorium endivia plant of the invention, which seedcontain in their genome the genetic information that encodes thereversible genic male sterility trait of the invention.

The invention also relates to the use of seeds that were deposited underNCIMB accession number 42092 for transferring reversible genic malesterility into another agronomically valuable endive plant.

The invention also relates to the use of a Cichorium plant, inparticular a Cichorium endivia plant of the invention that exhibitsreversible genic male sterility due to the presence, in the genome ofthe plant, of the reversible genic male sterility trait as found inseeds that were deposited under NCIMB accession number 42092 as a crop.

The invention further relates to the use of a Cichorium plant, inparticular a Cichorium endivia plant of the invention that exhibitsreversible genic male sterility due to the presence, in the genome ofthe plant, of the reversible genic male sterility trait as found inseeds that were deposited under NCIMB accession number 42092 as a sourceof seed.

The invention also relates to the use of a Cichorium plant, inparticular a Cichorium endivia plant of the invention that exhibitsreversible genic male sterility due to the presence, in the genome ofthe plant, of the reversible genic male sterility trait as found inseeds that were deposited under NCIMB accession number 42092 as a sourceof propagating material.

The invention also relates to the use of a Cichorium plant, inparticular a Cichorium endivia plant of the invention that exhibitsreversible genic male sterility due to the presence, in the genome ofthe plant, of the reversible genic male sterility trait as found inseeds that were deposited under NCIMB accession number 42092 forconsumption.

The invention also relates to harvested leaves of a Cichorium plant, inparticular a Cichorium endivia plant of the invention, to food productswhich may comprise harvested leaves of endive plants of the invention,either in natural or in processed form, and to a container which maycomprise one or more endive plants of the invention in a growthsubstrate for harvest of leaves from the endive plant in a domesticenvironment.

The invention further relates to the use of a Cichorium plant, inparticular a Cichorium endivia plant of the invention in breeding toconfer reversible genic male sterility.

The invention also relates to the use of the reversible genic malesterility trait as found in seeds that were deposited under NCIMBaccession number 42092 for conferring reversible genic male sterilityonto a Cichorium endivia plant.

The invention further relates to the use of a Cichorium plant as arecipient of the reversible genic male sterility trait as found in seedsthat were deposited under NCIMB accession number 42092, in which thegenetic determinant may be identified by a molecular markercharacterised by SEQ ID No. 2.

In one aspect the invention relates to a method for production of aCichorium plant, in particular a Cichorium endivia plant which exhibitsa reversible genic male sterility trait, which may comprise

-   -   a) crossing a plant which may comprise a genetic determinant        that leads to the trait with another plant;    -   b) selfing the resulting F1 for obtaining F2 plants;    -   c) selecting plants that have the trait in the F2;    -   d) optionally performing one or more additional rounds of        selfing or crossing, and subsequently selecting for a plant        which may comprise/showing the trait of the invention.

It is clear that the parent that provides the trait of the invention isnot necessarily a plant grown directly from the deposited seeds. Theparent may also be a progeny plant from the seed or a progeny plant fromseeds that are identified to have the trait of the invention by othermeans.

In one aspect, the invention relates to a method for production of aCichorium plant, in particular a Cichorium endivia plant, which exhibitsa reversible genic male sterility trait, which may comprise

-   -   a) crossing a plant which may comprise the genetic determinant        that leads to the trait with another plant;    -   b) optionally backcrossing the resulting F1 with the preferred        parent;    -   c) selecting for plants that have the trait in the F2;    -   d) optionally performing one or more additional rounds of        selfing or crossing, and subsequently selecting for a plant        which may comprise the trait.

The invention additionally provides a method of introducing anotherdesired trait into a Cichorium plant, in particular a Cichorium endiviaplant, which exhibits the reversible genic male sterility trait, whichmay comprise:

-   -   a) crossing a Cichorium plant, in particular a Cichorium endivia        plant, that exhibits the reversible genic male sterility trait,        representative seed of which were deposited under deposit number        NCIMB 42092, with a second Cichorium plant, in particular a        Cichorium endivia plant, that may comprise a desired trait to        produce F1 progeny;    -   b) selecting an F1 progeny that may comprise said reversible        genic male sterility trait and the desired trait;    -   c) crossing the selected F1 progeny with either parent, to        produce backcross progeny;    -   d) selecting backcross progeny which may comprise the desired        trait and the reversible genic male sterility trait; and    -   e) optionally repeating steps c) and d) one or more times in        succession to produce selected fourth or higher backcross        progeny that may comprise the desired trait and the reversible        genic male sterility trait. The invention includes a Cichorium        plant, in particular a Cichorium endivia plant, produced by this        method.

In one embodiment selection for plants exhibiting the reversible genicmale sterility trait is done in the F1 or any further generation byusing a molecular marker characterised by SEQ ID No. 2. In anotheraspect selection for the trait of the invention is started in the F2 ofa cross or alternatively of a backcross. Selection of plants in the F2may be done phenotypically as well as by using the said marker whichdirectly or indirectly detects the genetic determinant underlying thetrait.

Phenotypic selection may be done based on the observation of malesterility (whereby only non-functional pollen grains are formed) and/orbased on the observation of flower colour. When the genetic determinantunderlying the trait of the invention is present in a homozygous state,this correlates with a pale flower colour, when compared to plants nothaving the said genetic determinant, or to plants in which the saidgenetic determinant is present in a heterozygous state.

In one embodiment selection for plants exhibiting the reversible genicmale sterility trait is started in the F3 or a later generation.

In one embodiment the plant which may comprise the genetic determinantis a plant of an inbred line, a hybrid, a doubled haploid, or of asegregating population.

The invention further provides a method for the production of aCichorium plant, in particular a Cichorium endivia plant, exhibiting thereversible genic male sterility trait by using a doubled haploidgeneration technique to generate a doubled haploid line which maycomprise the said trait.

The invention also relates to a method for the production of a Cichoriumplant, in particular a Cichorium endivia plant, exhibiting thereversible genic male sterility trait by using a seed that may comprisea genetic determinant in its genome that leads to the reversible genicmale sterility trait for growing the said a Cichorium plant, inparticular a Cichorium endivia plant. The seeds are suitably seeds ofwhich a representative sample was deposited with the NCIMB under depositnumber NCIMB 42092.

The invention also relates to a method for seed production which maycomprise growing Cichorium endivia plants from seeds of which arepresentative sample was deposited under deposit number NCIMB 42092,reversing the reversible genic male sterility by the application ofjasmonic acid and/or one or more jasmonic acid derivatives, inparticular methyl jasmonate, allowing the plants to produce seeds, andharvesting those seeds. Production of the seeds is suitably done bycrossing or selfing. Crossing may e.g. be done by means of handpollination or by employing suitable pollinating insects.

In one embodiment, the invention relates to a method for the productionof a Cichorium plant, in particular a Cichorium endivia plant,exhibiting the reversible genic male sterility trait by using tissueculture.

The invention furthermore relates to a method for the production of aCichorium plant, in particular a Cichorium endivia plant, exhibiting thereversible genic male sterility trait by using vegetative reproduction.

In one embodiment, the invention relates to a method for the productionof a Cichorium endivia plant exhibiting the reversible genic malesterility trait by using a method for genetic modification to introgressin particular dominantly acting transgenes that cause the said traitinto the Cichorium endivia plant, for example by means of RNAi, amiRNAor antisense, or dominant-negative versions of a target gene, or throughmutation. Genetic modification may comprise transgenic modification ortransgenesis, using a gene from a non-crossable species or a syntheticgene, and cisgenic modification or cisgenesis, using a natural gene,coding for an (agricultural) trait, from the crop plant itself or from asexually compatible donor plant. The plant may also be geneticallymodified by mutation of a target gene to exhibit the male sterilitytrait of the invention.

The invention also relates to a breeding method for the development ofCichorium plants, in particular Cichorium endivia plants that exhibitthe reversible genic male sterility trait wherein germplasm which maycomprise said trait is used. Representative seed of said plant which maycomprise the genetic determinant and being representative for thegermplasm was deposited with the NCIMB under deposit number NCIMB 42092.

In a further embodiment the invention relates to a method for theproduction of a Cichorium plant, in particular a Cichorium endiviaplant, exhibiting the reversible genic male sterility trait whereinprogeny or propagation material of a plant which may comprise thegenetic determinant conferring said trait is used as a source tointrogress the said trait into another Cichorium plant, in particularanother Cichorium endivia plant. Representative seed of said plant whichmay comprise the genetic determinant was deposited under deposit numberNCIMB 42092.

The invention provides preferably a Cichorium plant, in particular aCichorium endivia plant, exhibiting the reversible genic male sterilitytrait, which plant is obtainable by any of the methods herein describedand/or familiar to the skilled person.

This invention also relates to the use of Cichorium plants exhibiting areversible genic male sterility trait as a female parent for thecreation of hybrid Cichorium seeds. The fact that Cichorium plants ofthe invention are unable to self-fertilise due to male sterility (ifthey are not rendered male-fertile by the application of jasmonic acidand/or one or more jasmonic acid derivatives, in particular methyljasmonate) makes it possible to efficiently use Cichorium plants of theinvention as female parents for the efficient creation of hybridCichorium plants, using pollen from other Cichorium plants. Pollinationof the plants of the invention may e.g. be achieved by means of manualpollination, or by employing suitable pollinating insects, such asbumblebees, flies or bees, and placing reversible genic male sterileCichorium plants of the invention alongside male-fertile Cichoriumplants that will function as father plants of the hybrid Cichoriumseeds, by providing functional pollen grains for a successfulfertilisation of the reversible genic male sterile Cichorium plants ofthe invention. This invention further relates to hybrid Cichorium seeds,obtainable by the use of Cichorium plants exhibiting a reversible genicmale sterility trait as a female parent.

This invention also relates to a food product, which may comprise edibleparts of plants of the Compositae family, optionally in processed form,wherein the plants comprise the genetic determinant underlying thereversible genic male sterility trait of the invention. These edibleparts may be leaves (e.g. in case of lettuce, endive, witloof andradicchio), or seeds (e.g. in the case of sunflower), or flowers (e.g.in the case of artichoke). The food products are preferably derived fromlettuce and endive.

The word “trait” in the context of this application refers to thephenotype of the plant. In particular, the word “trait” refers to thetrait of the invention, more in particular to the reversible genic malesterility trait. The term “genetic determinant” is used for the geneticinformation in the genome of the plant that causes the trait of theinvention. When a plant shows the trait of the invention, its genome maycomprise the genetic determinant causing the trait of the invention. Theplant thus has the genetic determinant of the invention. The term“genetic determinant” as used herein encompasses a gene or allele. Theseterms are used interchangeably. A genetic determinant may be identifiedby the use of a molecular marker. A genetic determinant mayalternatively be identified by the position on a genetic map, or byindication of the location on a linkage group or chromosome. When agenetic determinant is no longer linked to a specific molecular marker,but its position on a chromosome as defined on a genetic map isunaltered, this genetic determinant is still the same as when it waslinked to the molecular marker. The genetic trait that it confers istherefore also still the same.

The “genetic trait” is the trait or characteristic that is conferred bythe genetic determinant. The genetic trait may be identifiedphenotypically. However, also plant stages for which no phenotypic assaymay be performed do carry the genetic information that leads to thegenetic trait. “Trait” or “phenotypic trait” may be used instead of“genetic trait”.

It is clear that the parent that provides the trait of the invention isnot necessarily a plant grown directly from the deposited seeds. Theparent may also be a progeny plant from the seed or a progeny plant fromseeds that are identified to have the trait of the invention by othermeans.

This invention also relates to the use of plants of the Compositaefamily which may comprise the genetic determinant underlying thereversible genic male sterility trait of the invention, or of plantsproduced from the seed of such plants or from propagation material ofsuch plants, as germplasm in a breeding program for the development ofCompositae plants exhibiting a reversible genic male sterility that maybe reversed by the application of jasmonic acid and/or one or morejasmonic acid derivatives, in particular methyl jasmonate.

Seeds of Lactuca sativa which may comprise a genetic determinant of theinvention which leads to reversible genic male sterility that may bereversed by the application of jasmonic acid and/or one or more jasmonicacid derivatives, in particular methyl jasmonate, were deposited withNCIMB Ltd, Ferguson Building, Craibstone 5 Estate, Bucksburn, AberdeenAB21 9YA, UK on Oct. 8, 2012 under deposit accession number NCIMB 42060.All seeds of this deposit comprise the genetic determinant of theinvention in a homozygous state.

Seeds of Cichorium endivia which may comprise a genetic determinant ofthe invention which leads to reversible genic male sterility that may bereversed by the application of jasmonic acid and/or one or more jasmonicacid derivatives, in particular methyl jasmonate, were deposited withNCIMB Ltd, Ferguson Building, Craibstone 5 Estate, Bucksburn, AberdeenAB21 9YA, UK on 21 Dec. 2012 under deposit accession number NCIMB 42092.About 50% of the seeds of this deposit comprise the genetic determinantof the invention in a homozygous state, and about 50% of the seeds ofthis deposit comprise the genetic determinant of the invention in aheterozygous state.

Sequence Information

SEQ ID No.1: Genetic SNP marker that is able to identify the presence ofthe genetic determinant that underlies the trait of the invention inLactuca sativa plants grown from seeds that are deposited underaccession number NCIMB 42060. A genomic fragment of the lettuce OPR3gene is presented, wherein the position of the SNP which may comprise achange from C to T [C/T] is indicated as a T in bold and underlined.This SNP is present in plants grown from seeds of the said deposit.

atggctgaaacaccgccgtctgccgacaatccaactctcttttctccattcaagatgggcaagtttaatctctctcacaggtccgttgtttctatcctttcttctcttcactttctaatcataaatccgtcccccctataaaagtccttcagattttgagcttgatccagtcttgaccgatcactataccgtgttttggttgtagggtggtgttagctccgatgacgcggtgtagggcgttaaatagcataccgaatcaagctctggtggagtattacaggcagagagcaaccgccggtgggtttctcatcacggaggggacaatgatctctcctacctccgccgggtaatttcgctattccttttgttcttcaagggtgttttagtaaatcaacattccaacataattcaccgggacaaaccatataaaaccgccacgtggcaattcttagttccttacatagtgctttgtgggacctgcagtatagactattattaaagtcacaccattattaaagtcacattctctttataaccactttataaagtctttataactaaaaaatgtgtttttttcgttgtactttatagttagattagatgcataatgtggatcttatgaaccattaaacaatgatacaagctacttgtgttctaaagttaaagatgccattttgattcttaatttaaaaaacccatttggtttaggaatacccctttattcatcgatcattataaagcccaaattaacgatctttctggtaaaaaaaaacccaatttggtgatcaggttccctcacgtaccaggtatatttaatcaagaacaagttgaagcttggaagaaagtcgtggatgcagttcatgaaaaaggcgctgtgatcttttgt T aattatggcatgtcggcagagcatcccaccaaggtacgctttcttccatctaaaagtctcaaaatctcaacattttgatttttgaacctaaattcgaatcgaaagtgatcattgtgttgaacaaacagtatatcaacctaatggggttgcaccaatatcatctacaagcaaacccatatcgaaaaaatggagaattttaatgcccgatgggacccacgctcaatatccaaaccctcgaccactcgctacccatgaaataccagaggtggtggaagactatcgtctggcagcaattaacgccattgaagcaggttttgatggaatcgagattcacggagcccatggttatcttctcgatcaattcatgaaagatggcatcaataatcgaaccgatgaatatggtggatctttagcaaaccgatgcaaattcttactgaaagtggtgaaatcgatagctacagccattggtgcagataaagtcggtgttagaatctcaccagctattgaccatttagacgccatggattctgacccacgtagcttagggcttgaagtaattgaaagactgaataaacttcaggttgaattagggtcaaagttgacttatcttcatgtgactcaaccaaggtacacggcttatggtcaaacagaagctggaagccatggaagtgaagaggaagttgctgagttgatgaagatatggagaagggcatttatgggaacttttgtttgtagtggtgggtatactagagagcttgggattgaagctgtggctaaaggggatgctgatttggtggcttatggaaggctttttatatcgaatccggatttggttttgagactcaaggttaatgcacctttgaataggtatgttagggctagtttttatacacatgatcctgttgtagggtacactgattacccttcacttgagaA

SEQ ID No. 2: Genetic SNP marker that is able to identify the presenceof the genetic determinant that underlies the trait of the invention inCichorium endivia plants grown from seeds that are deposited under NCIMBnumber 42092. A genomic fragment of the endive OPR3 gene is presented,wherein the position of the SNP which may comprise a change from C to A[C/A] is indicated as A in bold and underlined. The SNP is present inplants grown from seeds of the said deposit.

TCAACATCCAGATCTCCGATCCCAAGCAAATAATGGCTGAAACGACGCCGTCTGCCGACAATCCAACCCTCTTTTCTCCGTACAAGATGGGCAAGTTCAATCTCTCTCACAGGGTGATCAGTTACTTTACTTCGATCCATTGTTCTCGCAGCTTTCTGATCATACTTCATTCCCCCAGCAAAATAATTCAGAATTTGACCTTGATCCAATTCTGTTTTTAATTCTTTTACCGATTAGTTCACCGTGTTTTGGTTGTAGGGTGGTGTTAGCTC A GATGACGAGATGCAGG

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined in the appended claims.

The present invention will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the invention in any way.

EXAMPLES Example 1 Reversible Male Sterility in Lettuce

A mutant lettuce plant (Lactuca sativa), having a mutated version of theOPR3 gene, was obtained by means of chemical mutagenesis and subsequentTILLING screening of about 3,000 M2 plants. DNA sequencing revealed thepresence of a C to T mutation (CAA to TAA) in one M2 plant, leading atthe protein level to the conversion of a Glutamine to a premature stopcodon at position 107, and hence to the presumed expression of atruncated version of the OPR3 protein in this mutant plant.

Phenotypically this mutant lettuce plant was indistinguishable from wildtype lettuce plants, when the mutation was present in a heterozygousstate. However, when the plant was self-pollinated and allowed toproduce seeds, the homozygous mutant plants of the next generationshowed complete male sterility. The few pollen grains that developed insuch homozygous mutants were not functional. The homozygous mutantplants did not set seed when allowed to self-pollinate, but when used asthe female parent in a cross, the plants were able to set seed, whichdemonstrated that their female fertility was not impaired. In thismanner the mutation could be maintained in subsequent generationsthrough screening of progeny plants of the selfing of a heterozygousmutant plant with a molecular marker designed to identify the presenceof the causal mutation, SEQ ID No. 1, even though the homozygous mutantplants were male sterile and failed to set seed throughself-pollination.

Surprisingly, it was observed that spraying young flower buds with a 200μM (0.2 mM) methyl jasmonate (MeJA) aqueous solution (in water plus0.025% Zipper surfactant) was sufficient to restore male fertility inthese homozygous mutants. From the early bolting stage theinflorescences were sprayed 5 times per week, for a total of 3 weeks. Incontrast to mock-treated control plants—that were sprayed with anaqueous solution of 0.025% Zipper surfactant—the MeJA treated plantsproduced seeds in a subset of their flowers, being the flowers that hadreceived an effective MeJA dose at the appropriate developmental stage(FIG. 1).

The seeds that were produced by the homozygous mutant plants treatedwith MeJA were all homozygous for the mutation, as could be demonstratedby use of a molecular marker detecting the causal mutation (SEQ ID No.1).

Example 2 Transfer of the Reversible Male Sterility Trait to AnotherLettuce Plant

Lettuce plants of the invention (of the deposit, NCIMB 42060) werecrossed with wild type (WT) lettuce plants, which do not carry the traitof the invention. The resulting F1 plants from this cross had the samephenotype as the WT plant, i.e. they displayed normal fertility.Nevertheless, the presence of the trait of the invention in aheterozygous state could be detected in all F1 plants by means of amolecular marker. The Single Nucleotide Polymorphism (SNP) that can beused for this purpose is presented as SEQ ID No. 1. This molecularmarker can be used to identify the presence of the genetic determinantthat underlies the trait of the invention in lettuce plants grown fromseeds as deposited under NCIMB number 42060.

In the F2 generation the trait of the invention segregated in a mannerthat corresponds with a monogenic recessive inheritance. The trait ofthe invention could be introduced into a wild type lettuce plant bycrossing the wild type plant with a plant of the invention and selectingfor the desired phenotype, by selection on male sterility and/or thepresence of the molecular marker in a homozygous state. The lattermethod of detection has the advantage that plants of the invention canbe identified at a young developmental stage, long before they startflowering. This enables the confident selection of desired plants(carrying the trait of the invention) at the seedling stage, whichallows for a more efficient use of plant growth facilities.

The wild type lettuce plant into which the trait of the invention can beintroduced can be a lettuce plant of any leaf type, any form or anycolour.

In the F2 generation the male sterility trait was also reversible. Theaddition of jasmonic acid and/or one or more jasmonic acid derivatives,in particular methyl jasmonate, was sufficient to restore malefertility, as described in Example 1.

Example 3 Reversible Male Sterility in Endive

A mutant endive plant (Cichorium endivia), having a mutated version ofthe OPR3 gene, was obtained by means of chemical mutagenesis andsubsequent TILLING screening of about 10,000 M2 plants. DNA sequencingrevealed the presence of a C to A mutation in exon 2 (CCG to CAG) in oneM2 plant, leading at the protein level to a Proline to Glutamine aminoacid change at position 76.

Phenotypically this mutant endive plant was indistinguishable from wildtype endive plants, when the mutation was present in a heterozygousstate. However, when the plant was self-pollinated and allowed toproduce seeds, the homozygous mutant plants of the next generationshowed male sterility, along with a pale flower colour (almost white,compared to the blue colour of wild type flowers of this endive line).The pollen grains that developed in such homozygous mutants were unableto germinate and therefore dysfunctional. When used as the female parentin a cross, the plants were able to set seed, which demonstrated thattheir female fertility was not impaired. In this manner the mutationcould be maintained in subsequent generations, even though thehomozygous mutant plants were male sterile and failed to set seedthrough self-pollination.

Surprisingly, it was observed that spraying young flower buds on a dailybasis—throughout the entire flowering stage of the plant, which lastedabout 3.5 months—with a MeJA solution had a dramatic effect on themutant flowers that developed from the treated flower buds (an aqueoussolution of 200 μM (0.2 mM) MeJA with 0.025% Zipper surfactant). Insteadof developing into flowers with a pale colour, as was the case foruntreated or mock-treated flower buds (treated with an aqueous solutionof 0.025% Zipper surfactant), the treated flower buds developed intoblue flowers. Blue was the wild type flower colour in this endivebackground. In addition, the flowers that developed from MeJA-treatedflower buds produced viable, functional pollen and were able to set seedthrough self-pollination, whereas control plants sprayed with an aqueoussolution of 0.025% Zipper surfactant remained male sterile and hencedevoid of seeds.

Other application methods (other than spraying) of jasmonic acid and/orone or more jasmonic acid derivatives, in particular methyl jasmonate,had comparable effects, such as “painting” a MeJA solution onto thesurface of young flower buds with a fine brush.

This experiment demonstrated that the male sterility that was caused bythe opr3 mutation in endive could be overcome by the application ofMeJA. The seeds that were produced by selfing of the homozygous mutantplants treated with MeJA were all homozygous for the mutation. Jasmonicacid or other derivatives thereof had the same effect.

The presence of the mutation could be detected by means of molecularmarkers (the SNP marker that can be used for this purpose is presentedas SEQ ID No. 2), and in flowering plants one could also use the flowercolour as a visual marker. Homozygous mutants have very pale (almostwhite) flowers than the flowers of wild type and heterozygous plants,which are blue (FIG. 2).

Example 4 Transfer of the Reversible Male Sterility Trait to AnotherCichorium Plant

The deposited endive seeds harbouring the trait of the invention(deposited as NCIMB number 42092) have been produced by allowing endiveplants homozygous for the reversible male sterility trait to bepollinated by bumblebees, with pollen from endive plants heterozygousfor the reversible male sterility trait. The latter plants are fullymale fertile. Of the seeds resulting from this cross about 50% werehomozygous for the trait of the invention, and about 50% wereheterozygous for the said trait.

Endive plants of the invention were crossed with wild type (WT) endiveplants, which do not carry the trait of the invention. Plants grown fromseeds of the deposit were first selected (phenotypically on the basis ofmale sterility and/or a pale flower colour, and/or on the presence ofthe causal SNP mutation in the OPR3 gene in a homozygous state), toensure that the trait of the invention would be transferred to the F1generation in 100% of the cases. Alternatively, this selection can bedone in the F2 generation, although this is less efficient.

The F1 plants resulting from this cross had the same phenotype as thewild type plant, i.e. they displayed normal fertility and a normal, blueflower colour. Nevertheless, the presence of the trait of the inventionin a heterozygous state could be detected by means of a molecularmarker. The SNP that can be used for this purpose is presented as SEQ IDNo. 2. This molecular marker can be used to identify the presence of thegenetic determinant that underlies the trait of the invention in endiveplants grown from seeds as deposited under NCIMB number 42092.

In the F2 generation the trait of the invention segregated in a mannerthat corresponds with a monogenic recessive inheritance. The trait ofthe invention could be introduced into a wild type endive plant bycrossing the wild type endive plant with an endive plant of theinvention and after selfing selecting for the desired phenotype in theF2 generation, for example by selection on male sterility and/or thepresence of the molecular marker in a homozygous state. The lattermethod of detection has the advantage that plants of the invention canbe identified at a young developmental stage, long before they startflowering.

In the F2 generation the male sterility trait was also reversible. Theaddition of jasmonic acid and/or one or more jasmonic acid derivatives,in particular methyl jasmonate, was sufficient to restore malefertility, as described in Example 3.

In a similar manner the trait of the invention can also be introducedinto e.g. witloof or radicchio plants (Cichorium intybus).

The invention is further described by the following numbered paragraphs:

1. Plant of the Compositae family exhibiting a reversible genic malesterility trait, characterised in that the genic male sterility iscaused by a reduction or complete absence of endogenous jasmonic acidproduction, resulting from interference with one or more target genesinvolved in endogenous jasmonic acid production, selected from the groupconsisting of lipoxygenase, allene oxide synthase, allene oxide cyclaseand 12-oxo-phytodienoic acid-10,11-reductase, or their functionalhomologues.

2. Plant of paragraph 1, wherein the male sterility can be reversed bythe application of jasmonic acid and/or one or more jasmonic acidderivatives, in particular methyl jasmonate.

3. Plant of paragraph 1 or 2, wherein the interfering with the one ormore target genes consists of preventing transcription thereof.

4. Plant of paragraph 3, wherein transcription is preferably preventedby means of RNA oligonucleotides, DNA oligonucleotides or RNAi moleculesdirected against the target gene promoter, or wherein transcription ispreferably prevented by means of the expression of a negatively actingtranscription factor acting on the target gene promoter.

5. Plant of paragraph 1 or 2, wherein the interfering with the one ormore target genes consists of destabilising the target gene mRNA ortranscript, preferably by means of nucleic acid molecules that arecomplementary to the target gene mRNA or transcript, selected from thegroup consisting of antisense RNA, RNAi molecules, Virus-Induced GeneSilencing (VIGS) molecules, co-suppressor molecules, RNAoligonucleotides or DNA oligonucleotides, or wherein the interferingwith the one or more target genes consists of inhibiting the target geneexpression product, preferably by means of the expression product(s) ofone or more dominant negative nucleic acid constructs, or preferably bymeans of one or more chemical compounds.

6. Plant of paragraph 1 or 2, wherein the interfering with the one ormore target genes consists of the introduction of one or more mutationsinto the target gene, leading to perturbation of its biologicalfunction, and wherein the one or more mutations are preferablyintroduced randomly by means of one or more chemical compounds, such asethyl methanesulphonate, nitrosomethylurea, hydroxylamine, proflavine,N-methyl-N-nitrosoguanidine, N-ethyl-N-nitrosourea,N-methyl-N-nitro-nitrosoguanidine, diethyl sulphate, ethylene imine,sodium azide, formaline, urethane, phenol and ethylene oxide, and/or byphysical means, such as UV-irradiation, fast-neutron exposure, X-rays,gamma irradiation, and/or by insertion of genetic elements, such astransposons, T-DNA, retroviral elements, and/or wherein the one or moremutations are introduced specifically by means of homologousrecombination or oligonucleotide-based mutation induction.

7. Plant of any one of the paragraphs 1-6, wherein the plant is a plantof the genus Lactuca.

8. Plant of paragraph 7, wherein the plant is a plant of the speciesLactuca sativa, and wherein the male sterility trait is caused by agenetic determinant, which is as present in the genome of plants grownfrom seed of which a representative sample was deposited with the NCIMBunder accession number NCIMB 42060, in which the presence of the saidgenetic determinant can be identified by a molecular markercharacterised by SEQ ID No. 1.

9. Plant of the Compositae family of any one of the paragraphs 1-6,wherein the plant is a plant of the genus Cichorium.

10. Plant of paragraph 9, wherein the male sterility trait is caused bya genetic determinant, which is as present in the genome of plants grownfrom seed of which a representative sample was deposited with the NCIMBunder accession number NCIMB 42092, in which the presence of the saidgenetic determinant can be identified by a molecular markercharacterised by SEQ ID No. 2.

11. Plant of paragraphs 9-10, wherein the homozygous presence of thegenetic determinant underlying the male sterility trait can beidentified by a pale flower colour, when compared to plants not havingthe said genetic determinant, or to plants in which the said geneticdeterminant is present in a heterozygous state.

12. Use of the plant of paragraphs 7-8 as a female parent for thecreation of hybrid Lactuca seeds.

13. Use of the plant of paragraphs 9-11 as a female parent for thecreation of hybrid Cichorium seeds.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

What is claimed is:
 1. A Lactuca sativa plant comprising a reversiblegenic male sterility trait, wherein the genic male sterility trait iscaused by a genetic determinant, which is as present in the genome ofplants grown from seed of which a representative sample was depositedwith the NCIMB under accession number 42060, wherein said geneticdeterminant is SEQ ID No:
 1. 2. A method of creating hybrid Lactucaseeds comprising pollinating the plant of claim 1 with a pollen of amale-fertile Lactuca sativa plant.